Fluorescent dyes have been used for the detection and analysis of biological samples. As fluorescent dyes are highly sensitive, they can be used to detect a very small number of fluorescent molecules. For example, such fluorescent dyes can be used to detect fewer than 50 fluorescent molecules that are associated with cells. Barak, et al., J. Cell Biol. 90, 595 (1981).
Fluorescent dyes may be used as probes for use in imaging in live cells or tissue samples. For example, a fluorescent-dye probe bound to a receptor on the surface of Dictyostelium cells has been used in the imaging of a single molecule of fluorescently labeled cAMP. Ueda, et al., Science 294, 864 (2001). Several fluorescent probes having different fluorescent wavelengths may be used to perform multi-color imaging in live cells or tissue samples. Fluorescent probes are highly sensitive, of relatively low toxicity, and easy to dispose of relative to radioactive probes.
Fluorescent dyes can be used in the detection of nucleic acids, including DNA and RNA, and biological samples involving nucleic acids. Nucleic acid polymers such as DNA and RNA are involved in the transmission of genetic information from one generation to the next and to the routine functioning of living organisms. Nucleic acids are thus of interest and the objects of study. Fluorescent nucleic acid dyes that specifically bind to nucleic acids and form highly fluorescent complexes are useful tools for such study. These dyes can be used to detect the presence and quantities of DNA and RNA in a variety of media, including pure solutions, cell extracts, electrophoretic gels, micro-array chips, live or fixed cells, dead cells, and environmental samples. These dyes can be used in the quantitative detection of DNA in real-time polymerase chain reaction (qPCR), which is a technique used in genomic research and medical diagnosis.
Polymerase chain reaction (PCR) is a primer extension reaction that provides a method for amplifying specific nucleic acids in vitro. Generally, in PCR, the reaction solution is maintained for a short period at each of three temperatures, 96° C., 60° C. and 72° C., to allow strand separation or denaturation, annealing, and chain extension, respectively. These three temperature stages are repeated for 30 or 40 cycles with the use of an automated thermo-cycler that can heat or cool the tube containing the reaction mixture very rapidly. By repeating the PCR cycle, a million-fold copies of a DNA sample can be produced in a single enzymatic reaction mixture within a matter of hours, enabling researchers to determine the size and sequence of target DNA. This DNA amplification technique has been used for cloning and other molecular biological manipulations. Further discussion of PCR is provided in Mullis, et al., Methods Enzymol. (1987), and Saiki, et al., Science (1985).
One PCR-based technique that is useful is quantitative real-time PCR (qPCR). Briefly, the mechanism of qPCR is based on PCR amplification of a target DNA in an exponential manner. By running a PCR reaction and measuring the total number of DNA copies at given points during the course of the amplification reaction, one can retroactively calculate the amount of starting DNA material.
Fluorescence-based DNA detection is a generally sensitive, versatile, and convenient detection method that is used in qPCR. There are two types of fluorescent reagents used in qPCR. The first type is based on oligonucleotides labeled with one or more fluorescent dyes, or with a combination of a fluorescent dye and a quencher dye. These labeled oligonucleotides release fluorescence either upon hybridization to a target sequence, or upon cleavage of the oligonucleotides following hybridization in a manner proportional to the amount of DNA present. The mechanism and the use of the oligo-based fluorescent reagents have been described in various patents and publications. See, for example, Holland, et al., Proc. Natl. Acad. Sci. USA (1991); Lee, et al., Nucleic Acids Res. (1993); and U.S. Pat. Nos. 5,210,015, 5,538,848, 6,258,569, 5,691,146, 5,925,517, 5,118,801, 5,312,728, and 6,635,427. Although oligo-based fluorescent reagents for qPCR have the advantage of being highly specific toward a target sequence, they are very complex in design and consequently expensive to use. The second type of fluorescent reagents used in qPCR is based on DNA-binding fluorescent dyes, which are commonly referred to as fluorescent nucleic acid dyes or stains. Because fluorescent nucleic acid dyes are relatively simple molecules, they are easy to manufacture and thus inexpensive to use. Their application in qPCR is useful for routine genetic detection in research labs.
Not all commonly available fluorescent nucleic acid stains can be used for qPCR. Ideally, a fluorescent nucleic acid dye should meet certain criteria for it to be suitable for qPCR use. First, it should be chemically stable during PCR and storage. Since PCR is carried out at high temperature, the dye should be thermo-stable. Additionally, since the pH of the Tris buffer used for PCR can vary considerably from alkaline (pH 8.5) at low temperature (4° C.) to neutral or slightly acidic at high temperature, the dye should be resistant to acid- or base-assisted decomposition. Second, the dye, when present in the PCR solution, should not inhibit the PCR process. Third, the dye should be non-fluorescent or minimally fluorescent in the absence of DNA, and should become highly fluorescent in the presence of DNA. Fourth, the dye should have absorption and emission wavelengths that are compatible with existing instruments, which are normally equipped with optical channels optimized for common fluorescent dyes, such as FAM, JOE, VIC (Applied Biosystems, Foster City, Calif.), TAMRA, ROX, Texas Red, Cy3, and Cy5, for example. Fifth, the dye should bind with DNA with little or no sequence preference. Sixth, the DNA-dye complexes should have fluorescence intensities that are linearly related to the amount of DNA present.
Given the foregoing criteria, it is not surprising that very few nucleic acid-binding dyes can be used for qPCR. Ethidium bromide (EB) is a DNA dye that has been used to demonstrate the feasibility of using a simple dye for qPCR. Higuchi, et al., Bio-Technol. 10(4), 413 (1992). However, EB suffers from problems of low sensitivity and undesirable wavelengths. A widely used dye for qPCR is SYBR Green I from Molecular Probes, Inc. (Eugene, Oreg. (OR)). Wittwer, et al., Biotechniques 22(1), 130 (1997). SYBR Green I is a cyclically substituted asymmetric cyanine dye. Zipper, et al., Nucleic Acids Res. 32(12), e103 (2004); and U.S. Pat. Nos. 5,436,134 and 5,658,751. The advantages of SYBR Green I are that it has excitation and emission wavelengths very closely matching those of FAM, with which most of the instruments are compatible, and that it is highly fluorescent when bound to DNA. Recently, a DNA dye called LC Green was used for qPCR, although the structure of the dye was not disclosed. Although the LC Green dye appears to have desirable wavelengths matching the commonly used FAM optical channel in most of the PCR instruments, it is much less sensitive than SYBR Green I. More recently, a DNA minor groove-binder called BEBO and a related dye called BOXTO, both of which are asymmetric cyanine dyes, have been reported for use in qPCR. Bengtsson, et al., Nucleic Acids Res. 31(8), e45 (2003); and U.S. Patent Application Publication No. 2004/0132046. Like LC Green, both BEBO and BOXTO significantly lag behind SYBR Green I in terms of sensitivity.
Although SYBR Green I has been widely used DNA dye for qPCR, it still is lacking in several respects. For one, SYBR Green I has an inhibitory effect on the PCR process, which limits the maximum signal strength one can achieve by increasing dye concentration. The fluorescent signal strength of qPCR using SYBR Green I is initially proportional to the dye concentration until the dye concentration reaches a point where the dye starts to inhibit the PCR process significantly. A further increase in dye concentration will actually lower the signal strength or increase the cycle number (Ct) because of reduced DNA amplification. For another, SYBR Green I is chemically unstable under alkaline conditions, such as the alkaline condition of the PCR buffer when stored at low temperature. It has been reported that SYBR Green I stored in Tris buffer at 4° C. decomposes significantly over the course of a few days and that the dye decomposition products are apparently potent inhibitors. Karsai, et al., BioTechniques 32(4), 790 (2002). For yet another, SYBR Green I provides only one fluorescence color. Many commercially available fluorescence detection instruments have multiple optical channels (the FAM optical channel and additional other optical channels) and are thus capable of detecting multiple fluorescence colors.
Development of fluorescent dyes or the making or the use thereof is desirable.